Anticancer Action of Xiaoxianxiong Tang in Non-Small Cell Lung Cancer by Pharmacological Analysis and Experimental Validation

Xiaoxianxiong Tang (XXXT) is a well-known traditional Chinese medicine formula. Evidence is emerging supporting the benefits of XXXT in ameliorating therapy for non-small cell lung cancer (NSCLC). The purpose of this study aimed to explore the effects and mechanisms of XXXT through network pharmacological analysis and biological validation. TCMSP database was used to identify potentially active compounds in XXXT with absorption, distribution, metabolism, excretion screening, and their potential targets. The disease targets related to NSCLC were predicted by searching for Therapeutic Target database, GeneCards database, DrugBank database, and DisGeNET database. Of the 4385 NSCLC-related targets, 156 targets were also the targets of compounds present in XXXT. Subsequently, GO function and KEGG pathway enrichment and PPI network analyses revealed that, of the 95 targets and 20 pathways influenced by 20 ingredients in XXXT, 20 targets were associated with patient survival, and XXXT could exert an inhibitory action on the PI3K-AKT signaling pathway. Moreover, XXXT restrained the proliferation of A549 and H460 cells in a concentration-dependent manner and suppressed the mRNA and protein levels of key targets CCNA2, FOSL2, and BIRC5 closely linked to the PI3K-AKT pathway. Hence, XXXT has the potential to improve therapy for NSCLC by targeting the PI3K-AKT signaling pathway.


Introduction
Lung cancer is the major cause of cancer-related deaths all over the world, which lies behind almost one-quarter of all cancer deaths [1,2]. Non-small cell lung cancer (NSCLC) is the main subtype of lung cancer, and its proportion is approximately 80% in all lung cancer cases. e propensity for recurrence and distant metastasis of NSCLC leads to poor prognosis [3]. It is worth noting that recurrence and metastasis also occur following target therapy and recent immunotherapy [4,5]. Moreover, some conventional therapies are accompanied by serious side effects, thus reducing the life quality of patients [6]. erefore, it is necessary to explore novel drugs for the treatment of NSCLC.
TCM has been employed for fighting cancer for thousands of years [7,8]. Xiaoxianxiong Tang (XXXT) is a prescription that was first recorded in the Treatise on Exogenous Febrile Disease (Shanghan Lun) in the Eastern Han Dynasty 1700 years ago. XXXT is composed of 3 herbs, including Coptidis Rhizoma (Huanglain in Chinese), Arum Ternatum unb (Banxia in Chinese), and Trichosanthes Kirilowii Maxim (Gualou in Chinese). Traditionally, XXXT was used to dissipate phlegm and reduce lumps. Recently, it has been reported that XXXT or its active ingredients exert antiproliferative effects on a variety of tumor cells [9][10][11][12][13][14]. However, the active components of XXXT and their molecular mechanisms on NSCLC are yet to be determined.
In order to understand the molecular mechanisms of XXXT in the anticancer action in NSCLS, we used network pharmacology to identify the active components present in XXXT and their potential targets. By interrogating the disease targets related to NSCLC, a pool of common targets was revealed and validated experimentally. e overall structure flowchart is shown in Figure 1.

Predication of Potential Targets of XXXT.
e bioactive compositions in XXXT were obtained from the Traditional Chinese Medicine System Pharmacology Database (TCMSP, https://tcmspw.com/index.php) [15], which captures the associations between herbs, targets, and diseases. Pharmacokinetic properties were known as the key parameters affecting biological activity, including absorption, distribution, metabolism, and excretion. Drug-likeness (DL ≥ 0.18) and oral availability (OB ≥ 30%) were applied to filter active compounds in XXXT, which were used to establish a database of target genes. e gene names were standardized and annotated through UniProtKB database (https://www. uniprot.org/) [16].

Gene Ontology and KEGG Pathway Enrichment Analyses.
DAVID (https://david.ncifcrf.gov/) [22], an online bioinformatics database, was widely applied to obtain the biological processes, cellular components, molecular functions, and KEGG pathways of drug and disease targets. GO and pathway terms were screened with a false discovery rate (FDR) <0.05 and ranked according to their count, and the top 20 GO/KEGG enrichment was further analyzed.

Network Construction and Topology Analysis.
e Cytoscape version 3.7.2 was applied to build the drug-disease-target network. In detail, the XXXT chemical ingredients and putative therapeutic targets against NSCLC were imported into the Cytoscape software, and the size of nodes was arranged according to the ascending order of the number of degree values. e Cytoscape was employed to map the network relationship of bioactive compounds, potential targets, and KEGG pathways for XXXT treatment of NSCLC. Topological properties of the network, including closeness, degree, and betweenness were calculated using the network analyzer tool within Cytoscape 3.7.2 and also were used to seek the main active ingredients and related targets of XXXT against NSCLC.

Screening of Core Targets of XXXT against NSCLC.
e Gene Expression Profiling Interactive Analysis (GEPIA, http://gepia.cancer-pku.cn/detail.php) [23] is an interactive RNA-Seq database for gene analysis and cancer type analysis. e targets in compound-target-pathway network were selected in the GEPIA server by "overall survival," and the cutoff values were chosen by "median" group. P < 0.05 and P (HR) < 0.05 were set as the significance threshold in "LUAD" datasets to filter core targets of XXXT against NSCLC.

Cell Lines and Drugs.
e lung cancer cell lines A549 and H460 were obtained from the National Collection of Authenticated Cell Cultures (Shanghai, China) and grown in a humidified incubator with 5% CO 2 at 37°C. All cells were cultured in RPMI 1640 medium supplemented with 10% v/v fetal bovine serum (AusGeneX) and penicillin-streptomycin antibiotic (100 ug/mL). XXXT is composed of Huanglain (Coptidis Rhizoma), Banxia (Arum Ternatum unb.), and Gualou (Trichosanthes Kirilowii Maxim). e above herbs were purchased from the Huayu Pharmacy Company (Shanghai, China) and XXXT stock was performed as described previously [24]. e vacuum-dried XXXT powder was dissolved in DMSO as stock with a concentration of 500 mg/mL and kept at 4°C.

Cell Counting Kit-8 Assay Assessed Cell Proliferation.
e Cell Counting Kit-8 (CCK8) assay (Sangon Biotech, Shanghai, China) was applied to assess cell viability followed by the manufacturer's instructions. A549 and H460 cells were cultured in a 96-well plate at a density of 2 × 10 3 cells/ well with RPMI-1640 medium with various concentrations (0.0625 mg/ml, 0.125 mg/ml, 0.25 mg/ml, 0.5 mg/ml, 1 mg/ ml) of XXXT for 72 h. CCK-8 solution were added directly to the 96-well plate (10 ul/well) and kept in the incubator for 2 h. Optical density (OD) 450 values were measured using a spectrophotometer ( ermo Fisher Scientific, Vantaa, Finland). Inhibition ratio (%) � (1−OD sample /OD control ) × 100%. e IC 50 value was calculated by using the sigmoidal doseresponse function in GraphPad Prism 8.0 software.   ermo Fisher Scientific Inc.) was used to perform RT-qPCR on the thermocycler (Applied Biosystems 7300; ermo Fisher Scientific Inc.). e mRNA expression levels were measured by the 2-ΔΔCq calculation method, and β-actin was utilized as an internal control. All primer sets are shown in the supplementary file (Supplementary Table 1).

Statistical
Analysis. GraphPad Prism 8.0 software was used for statistical analyses and graphs. e data in the in vitro were shown as means ± standard deviation (SD). Oneway ANOVA followed by Bonferroni test was performed to investigate the differences between groups. In all comparisons, differences were considered statistically significant when P < 0.05.

Active Compounds and Target Screening and PPI Networks Construction.
rough retrieving the TCMSP database and literature, a total of 72 effective components of XXXT were obtained, including 48 species of Coptidis Rhizoma, 13 species of Arum Ternatum unb, and 11 species of Trichosanthes Kirilowii Maxim. After ADME screening with the filtering criteria of OB ≥30% and DL ≥0.18, 33 candidate ingredients were considered as active components of XXXT (Table 1) and 210 putative targets were predicted (Supplementary Table 2). e disease targets related to NSCLC were predicted by searching for GeneCards, DrugBank, TTD, and DisGeNET databases, and 4385 targets were obtained after removing duplicated targets (Supplementary Table 3). e 210 targets of XXXT were mapped to 4385 disease targets related to NSCLC. e 156 common targets related to NSCLC and XXXT were obtained and treated as potential targets of XXXTagainst NSCLC (Figure 2(a), Supplementary Table 4). e protein-protein interaction was constructed by importing 156 common targets related to NSCLC and identified as XXXT into the STRING database. e Cytoscape was used to carry out the visual composition. e network includes 137 round nodes and 573 edges, which represented the interaction between potential protein targets and function (Figure 2(b)).

GO and KEGG Pathway Enrichment Analyses.
e above 156 targets were imported into the DAVID online server for GO and KEGG pathway analysis. In molecular function, enrichment information includes protein binding, enzyme binding, and chromatin binding (Figure 3(a)). Only 10 terms were selected with FDR <0.05 in cellular components and the GO terms ranked according to their counts, including the nucleus, cytoplasm, cytosol, and nucleoplasm ( Figure 3(b)). In biological processes, the targets were enriched in the inflammatory response, apoptotic process, response to hypoxia, angiogenesis, and cell proliferation (Figure 3(c)). KEGG pathway enrichment information indicated that the 156 common targets contributed to 60 pathways (FDR <0.05). Specifically, these pathways were mainly correlated with cancer, such as pathways in cancer, PI3K-AKT, MAPK, HIF-1, and TNF signaling pathway. As shown in Figure 3(d), the top 20 pathways ranked by count were closely related to the etiopathogenesis of NSCLC.

Topological Network Construction of XXXT against NSCLC.
e drug-disease-target network was constructed which contained 344 edges and 188 nodes ( Figure 4). e network revealed the relationship between 32 active ingredients and related 156 common targets (HL10, one of 33 active ingredients, was eliminated due to lack of a common target with NSCLC). An active compound could target different gene targets, and a gene target could be relevant to different active compounds, reflecting the multicomponent and multitarget characteristics of XXXT. e Cytoscape version 3.7.2 was used to establish a drug-target-pathway network of XXXT against NSCLC based on the KEGG pathway enrichment analyses. e network diagram revealed that the 20 active ingredients in XXXT acted on 95 targets and regulated 20 pathways to treat NSCLC. And, the interaction network comprised 135 nodes and 668 edges ( Figure 5).

e Cytotoxic Effects of XXXT on Lung Cancer Cells.
To verify the findings from the network pharmacological analysis, we investigated the cytotoxicity of XXXT to decrease the survival of the lung cancer cell lines. Various concentrations of XXXT (0-1 mg/ml) were used to treat two NSCLC cancer cell lines for 72 h, and cell viability was detected by CCK8 assay. As shown in Figure 7(a), XXXT treatment significantly reduced the cell viability of A549 and H460 cells compared with control, and the decrease in cell viability was dependent on the concentrations of XXXT. e IC 50 values were about 408 ug/ml in H460 cells, and 188 ug/ ml in A549 cells.        Table 5). In A549 cells, the mRNA levels of MMP3, RELA, and SERPINE1were upregulated, and the mRNA level of BIRC5, CCNA2, CYCS, FOSL2, NFATC1, and SPP1 was downregulated (Supplementary Table 6). We further detected the protein expressions of CCNA2 and FOSL2, and XXXT also significantly downregulated the protein levels of CCNA2 and FOSL2 in H460 and A549 cells (Figures 7(b)-7(e)).

Discussion
Due to the complexity and difficulty, the mechanism of the researches on Traditional Chinese Medicine formula has been limited. e network pharmacology is a useful biological information tool for method to analyze the underlying active compounds and give clues for potential mechanism of Traditional Chinese Medicine formula. Firstly, through ADME screening, 33 active compounds were identified from the three herbs in the XXXT, 11 in  [25]. Berberine increased the expression of caspase-3 and impaired mitochondrial membrane potential to induce cell apoptosis in human gastric cancer cells [26]. Another study has revealed that Berberine induced cell cycle arrest and inhibited migration and invasion of lung cancer cells [27]. Baicalein (BX3, MOL002714, OB � 33.52%, DL � 0.21), a flavone present in another ingredient from Arum Ternatum unb induced mitochondrial-dependent apoptosis, and blocked S-phase cell cycle in human cisplatin-resistant pancreatic carcinoma cell line CAPAN-2 [28]. Baicalin was known as the important component in several herbs, unleashing significant antiproliferative action in a range of cancer cell lines by modulating apoptosis and cell cycle [29]. e natural flavonoid diosmetin (GL2, MOL002881, OB � 31.14%, DL � 0.27) present in Trichosanthes Kirilowii Maxim exerts anticancer effects through apoptosis induction and G2/M cell cycle arrest in HepG2 cells [30]. Diosmetin also has proapoptotic activities against breast cancer cells via activating the mitochondria-mediated apoptotic pathway and inducing cell cycle arrest [31]. As mentioned above, 33 potential bioactive compounds in XXXT against    Evidence-Based Complementary and Alternative Medicine NSCLC were screened out by TCMSP-based systems pharmacology. Based on the above active compounds in XXXT, the further analysis of related core targets was conducted and confirmed. As XXXT has been reported to be effective on lung cancer, the potential targets of XXXT based on the above active compounds were overlapped with NSCLCrelated targets. en, combined with the KEGG and survival analysis results, 20 of candidate targets were selected and proceed to be proved. It was confirmed that XXXT could inhibit the mRNA and protein levels of CCNA2 and FOSL2 in vitro. CCNA2 is a regulator of the G1/S and G2/M transition, which targeted by coniferin (BX8). CCNA2 is highly expressed in several cancer cells and contributes to epithelial-mesenchymal transition through dual activation of WNT and PLC pathways [32,33]. FOSL2 targeted by baicalein, is a regulator of cell proliferation, differentiation, and transformation. Several studies have reported that the phosphorylation and upregulation of FOSL2 enhance tumor growth and invasion in A549 cells through miR-638 [34][35][36][37]. In A549 cells, the mRNA level of BIRC5 was downregulated, and XXXT treatment tended to decrease the levels of BIRC5 mRNA in H460 cells, though there was no statistically significant difference. BIRC5, an inhibitor of the apoptotic gene family, was targeted by quercetin (HL7).
e recent study noted that the high-expression BIRC5 was correlated with low overall survival in lung adenocarcinoma patients, and the overexpression of BIRC5 is a risk factor for a worse prognosis [38]. Apoptosis repeat protein domain was inhabited by BIRC5 to militate against apoptosis inhibition [39], and siBIRC5 could overcome the antiapoptosis protection of cisplatin-resistant cells [40]. As a necessary protein of CPC, BIRC5 is also required for the mitotic exit and plays a major role in the cell cycle [41].
Finally, the potential mechanisms of XXXT on NSCLC treatment were mapped by the herb-compound-targetpathway relationship diagram. Network pharmacological analysis uncovered that the 20 compounds XXXT ameliorate NSCLC therapy by regulating 19 pathways, such as PI3K-AKT, TNF, and MAPK signaling pathways. Further   Figure 5: Compound-target-pathway network of XXXT. e yellow ellipse nodes are the active compounds, the green rectangle nodes are the related target genes, and the purple arrow nodes are the pathway. 10 Evidence-Based Complementary and Alternative Medicine   experiments in vitro revealed that the potential targets of XXXT for NSCLC treatment are closely related to the PI3K-AKT pathway. As a master regulator, PI3K-AKT signaling pathway influences cancer cell proliferation, metastasis, and metabolism [42][43][44]. e suppression of PI3K-AKT signaling pathway is an attractive strategy for NSCLC management via promoting apoptosis and inhibiting cell growth and invasion [45]. Apoptosis and cell cycle are two vital biological processes, by which the PI3K signaling pathway regulates tumor cell proliferation and viability [46][47][48]. Apoptosis and cell cycle arrest can be induced by several physiological regulations and chemical stimulation, the induction of which is recognized as effective steps for the treatment of cancer [49][50][51].
erefore, we suggest that XXXT can inhibit the PI3K-AKT signaling pathway in NSCLC by targeting CCNA2, FOSL2, and BIRC5 ( Figure 8).  Figure 6: e herb-compound-target-pathway relationship diagram to depict the mechanism of XXXT against NSCLC. e rectangles from left to right represent the herbs, active compounds, core targets, and KEGG pathways of XXXT against NSCLC.

Conclusion
For the study, network pharmacology was employed to investigate the drug-component-target-disease interaction of XXXT in the treatment of NSCLC. XXXT may induce lung cancer cell apoptosis and cell cycle arrest by regulating the PI3K-AKT signaling pathway. e results from this study provide insight into anticancer action and potential mechanism of XXXT against NSCLC and lay the foundation for further development of XXXT or its active ingredients as the complementary therapy for NSCLC treatment.  Figure 7: e cytotoxic effects of XXXT on lung cancer cells, RT-qPCR array, and western blot for core targets. (a) e inhibition rates of H460 and A549 cells treated with XXXT (0-1000 μg/mL) for 72 h were determined using CCK8 assay. A549 or H460 treated with XXXT at IC 50 for 72 h were harvested for analysis (b-e). (b) Heatmap of 20 core targets mRNA expression in the control group and XXXT treatment group. e significant difference of core targets in A549 and H460 cells (c) was observed, including expression of CCNA2 and FOSL2 mRNA. e protein levels of CCNA2 and FOSL2 were determined by western blot (d). Statistical analysis of CCNA2 and FOSL2 proteins expression intensity (e). e above data are presented as mean ± SD for three independent experiments. NS, not significant, * P < 0.05 and * * P < 0.01 showed significant difference vs. the control group.

Supplementary Materials
All primer sets in the RT-qPCR array are shown in the Table 1. Table 2 indicates the targets in XXXT. Table 3 indicates the targets related to NSCLC. Table 4 indicates common targets of NSCLC and XXXT. Table 5 indicates the result of the RT-qPCR array in H460 cells. Table 5 indicates the result of the RT-qPCR array in A549 cells. (Supplementary Materials)